***To ensure that no extra mutations are introduced in the vector backbone, site-directed mutagenesis strategies that use whole vector amplification require subcloning of the sequenced insert into a vector that hasn’t been amplified by PCR. Alternatively, the whole vector can be sequenced.

TransBionovo's Fast Mutagenesis System

Advantages

In vitro digestion and in vivo degradation of non-mutated parental DNA.

95% of clones contain the desired mutation.

Suitable for insertions of ∼ 45 bases.

Suitable for large deletions.

Easy 3-step workflow***.

Disadvantages

Long PCR protocol – 25 cycles (between 4 and 8 hours).

Partially complementary primers may anneal together.

Tandem primer insertion at or near the mutated site may occur.

Large deletions may be challenging because of the partial overlap between primers.

***To ensure that no extra mutations are introduced in the vector backbone, site-directed mutagenesis strategies that use whole vector amplification require subcloning of the sequenced insert into a vector that hasn’t been amplified by PCR. Alternatively, the whole vector can be sequenced.

Disadvantages

Only 1 primer contains the mutation which may generate non-methylated and non-mutated PCR products.

Primers or Dpn I-generated fragments are likely to be inserted at the ligation site.

***To ensure that no extra mutations are introduced in the vector backbone, site-directed mutagenesis strategies that use whole vector amplification require subcloning of the sequenced insert into a vector that hasn’t been amplified by PCR. Alternatively, the whole vector can be sequenced.

Disadvantages

***To ensure that no extra mutations are introduced in the vector backbone, site-directed mutagenesis strategies that use whole vector amplification requires subcloning of the sequenced insert into a vector that hasn’t been amplified by PCR. Alternatively, the whole vector can be sequenced.